Project Summary The ability to sense and respond to complex environmental signals such as light, oxygen and nutrients is critical for survival and adaptation of living organisms. Many signaling proteins adopt multi-domain modular architecture to accomplish the perception of input signals and the generation of an output biological response within the same protein molecule. One example of widespread modular systems is offered by bilin-based photoreceptors in the phytochrome superfamily. Since light can readily penetrate the cell membrane, these soluble multi-domain photoreceptors offer excellent model systems for studying the still elusive mechanism of long-range signaling and allosteric regulation in modular signaling proteins such as chemoreceptors and mechanoreceptors. Our long-term goal is to understand how modular photoreceptors perceive, integrate, and transduce signals at the molecular level. To attack this goal, we adopt an integrated approach of biochemistry, spectroscopy, crystallography and cryoEM single particle reconstruction, with a main thrust on dynamic crystallography, which enables direct observation of structural responses at atomic resolution. In this proposal, we use two dual-sensor photoreceptors to represent two major types of bilin-binding photoreceptors. Both are sensory histidine kinases that feature different color perception and distinct signaling logic in response to light or chemical signals. Previously, we have obtained abundant structural information on various isolated domains by both static and dynamic crystallography. In this proposal, we will investigate the molecular mechanisms of signal integration and allosteric activation in full-length proteins where the sensor and effector domains are coupled. Specifically, we will capture structural changes in each sensory site by introducing perturbations via ligand soaking and light illumination. We will examine how the structural signals are initiated and how they propagate through the protein framework. We will jointly analyze the structures determined in different signaling states to dissect subtle motions that may involve bending, torque, winding/unwinding, or longitudinal sliding of helices. We will also perform mutagenesis and kinase assays to identify the key structural elements responsible for signal coupling between the sensor domains, the helical spine and the effector domain. We will determine the structures and dynamics of full-length photoreceptors by complementary approaches of crystallography and electron microscopy to address whether the structural asymmetry of the sensor and effector domains tethered in the same dimer scaffold plays an important role in allosteric regulation of modular photoreceptors. Our results will not only apply to photoreceptors but will inform the more general principles by which multi-domain signaling proteins such as the more widely studied chemoreceptors detect and process complex environmental signals at the molecular level. Use of light as operands in arithmetic and logic operations that override, negate, or modulate a desired cellular response is of great importance both for basic science and potentially for biotechnology applications.